Cas9 & Cpf1 secretion
There are also some things in the OUTCASST toolkit that need to be changed in comparison to the experimental approach in order to prepare the system for diagnosing Chagas disease. One of these things is the use of HEK293T cells, which need a very stable environment to stay alive. In the eventual tool, we will need to use cells that are more resistant to environmental fluctuations yet still cannot survive outside of the device (Patrick van Zon and Pieter-Jaap Krijtenburg, University Medical Center of Utrecht: genome diagnostics.) We also used a fluorescence signal as output in the experiments, which requires a fluorescence microscope to analyse the test results. To avoid the need of these and other equipment, we would ideally use an output signal in the form of visible light or, more promisingly, a change of color that is visible to the naked eye. Another thing we should keep in mind is the time it takes to get the results from our test device (Marit de Wit, Doctors without Borders).
There are also technical aspects that should be considered, like the method used for lysis of the parasites in the sample. Lysis needs to occur to get free DNA, i.e. a hypotonic solution (Jaap van Hellemond, Erasmus University Medical Center Rotterdam: parasitology). If a colorant is used as reporter mechanism, we need to remove the red color of heme groups from red blood cells, too, as it would interfere with the output signal.
Lastly, we should consider the target DNA we want to use to detect the parasites. Things that require careful consideration are GC-content, which has influence on binding affinity and specificity of the guide RNA. Specificity needs to be mutation specific as a strand with different base pairs should, ideally, not activate the system (Hans Bos and Hugo Snippert, University Medical Center Utrecht: cancer research).
Toolkit design solutionsThe OUTCASST toolkit has a closed box design, wherein all the components to perform the test are present in distinct compartments, separated by seals. These seals can be broken by applying pressure on them.
As was stated earlier, a lot of variables need to be kept constant to keep the HEK293T cells alive. Because of this, it is not feasible to use these cells in our design. Instead, we opt to use air-dried cells from the anhydrobiotic insect, Polypelidum vanderplanki, which can be stored at room temperature for 251 days and can restart proliferating again after rehydration 1. This way the shelf life of our tool can also be prolonged. To prevent the risk of our GMO getting out in the environment, several mechanisms and kill-switches will be incorporated in the cells, so they can only survive in our closed box system, in their resurgent state. This can be done by manipulating the metabolism, so that the cells can’t produce a crucial substance for survival, e.g. an amino acid, which will be added in the toolkit medium. In case the cells get out of the toolkit, they will die because of the absence of the crucial substance.
Rehydration can be done with a suitable medium. This has to be done one hour before use. The seal between the dried insect cells and the medium can be broken to pump the medium manually to the cells. After rehydration, the medium can be manually pumped to the waste compartment.
The next step is to add the two guide RNA’s to the revived cells. The gRNA’s are present in the design as dry powder to prevent premature degradation. This time, two seals need to be broken. First, the gRNA needs to be dissolved with the contents of another medium compartment. Then the medium with gRNA can be pumped to the cells where they will bind to dCas9 and dCpf1 on the extracellular cell membrane. This process takes about 10 minutes and after that, the medium with excessive gRNA can also be pumped to the waste compartment.
These are the preparation steps before the real diagnosis can start. First off, a blood sample has to be taken from a patient that might be infected with Chagas disease. To prevent the blood from clotting, heparin or EDTA can be added to the sample. The blood sample can then be introduced to the tool, after which the device needs to be sealed. To get access to the parasite DNA, all cells need to be lysed, including the red blood cells. This is done with a lysis buffer, a hypotonic solution.
The next step is to pump everything to a next compartment, wherein there are heme-binding compounds (such as HEBP) linked to the inside surface to decolorize the sample. Then a hypertonic resetting buffer is added to return the sample to isotonic levels, in order to prevent damage to the detector cells.
Now, the seal separating the sample from the cells can be broken and the sample can be introduced to the actual sensor. A color signal will appear after about 10 to 12 hours in case the patiënt is infected and will continue to become more visible after that.
The output signal will be a blue chromoprotein. This way, the sample color will become blue (or purple if there is still a little bit of heme in the sample) upon detection of the targeted DNA sequence.
After use, the tool should be disposed of in a safe manner preventing it to end up in the environment. Therefore, there will be a disposal guideline added in the toolkit manual. The test can be disposed of in a self-sealing bag, which can be boiled after the test is completed to minimize the risks.
Additional considerationsThere are still a lot of things that should be considered to make the OUTCASST tool optimal for diagnosing Chagas disease.
The first thing we still need to consider is the blood sample size needed to perform the test. From the patients aspect it would be best to take as little as possible. A smaller blood sample would also mean that the device can be made smaller, which in turn also makes the production costs for one test cheaper. However, there needs to be enough pathogen DNA in the blood sample to make sure that the test gives the right results. It would be possible to pretreat a larger sample to concentrate it before applying, increasing the chance of correct diagnosis, but this would again require skilled professionals and materials.
We have also thought about a question that was raised at the University Medical Center at the Cancer department. The question was why we wanted to express our system on the membrane of eukaryotic cells and not just express it intracellularly in bacteria. Then a blood sample could be added and the bacteria can be heat shocked to get the pathogen DNA intracellular, activating the binding of the two proteins. In this case, there would be a loss of the amplification step, since the transcription factor is then able to activate the reporter gene without a signal or cleavage of the transcription factor. Since we don’t know what the minimum amount of blood needed is, we wanted to design it in the way we can get the most signal, which is to include the amplification step. If it would prove that this amplification step is not needed, we could also just put the proteins in the tool and use a split reporter. On the other hand, the tool would not rely on use of living cells, which would make the use of our tool a whole lot safer.
We should also consider the material that the device is going to be made of. It should be of sturdy quality to prevent contamination of the environment with the device’s content. From the production perspective, the costs to produce it should be as low as possible to make the tool affordable. A main issue with costs, currently, is the production of the gRNA as it is expensive to synthesize.
We have also heard that the tool should have a low incidence of false positive and negative results and that our device should distinguish DNA strands with one different base pair. We want to take this information into account to design the target DNA. There are two possibilities from which we can choose. The first option would be to permit certain mutations in the target DNA, to prevent getting a false negative result in some cases. The second option would be to use a very conserved domain as target DNA and don’t allow any mismatches. From our perspective, we think the second option would be more suitable, since the specificity in our system is a very valuable aspect of the design. We have chosen to use the satellite DNA, which is present in the T. cruzi parasite as a 195 base pair repeat with about 100,000 copies (Aldert Bart, Academical Medical Center Amsterdam: Clinical molecular parasitologist).